1. Field of the Invention
The present invention relates to the use of immunological reagents specific for a human transmembrane efflux pump protein (P-glycoprotein) in a biochemical conformation adopted in the presence of certain cytotoxic, lipophilic drugs that are substrates for P-glycoprotein, in the presence of cellular ATP depleting agents, and by certain mutant embodiments of Pgp. The invention provides such immunological reagents for immunodiagnostic and therapeutic uses, for isolating lymphocytes and hematopoietic stem cells, and for anticancer drug development.
2. Background of the Invention
Many human cancers express intrinsically or develop spontaneously resistance to several classes of anticancer drugs, each with a different structure and different mechanism of action. This phenomenon, which can be mimicked in cultured mammalian cells selected for resistance to certain plant alkaloids or antitumor antibiotics such as colchicine, vinblastine and doxorubicin (former known as Adriamycin), is generally referred to as multidrug resistance ("MDR"; see Roninson (ed)., 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in Biochemical Bases for Multidrui Resistance in Cancer, Academic Press, N.Y., Chapter 11 for reviews). The MDR phenotype presents a major obstacle to successful cancer chemotherapy in human patients.
MDR frequently appears to result from decreased intracellular accumulation of drug as a consequence of increased drug efflux related to alterations at the cellular plasma membrane. When mutant cell lines having the MDR phenotype are isolated, they are found to express an ATP-dependent non-specific molecular "pump" protein (generally known as P-glycoprotein) that is located in the plasma membrane and keeps the intracellular accumulation of an anti-cancer drug low enough to evoke the drug-resistance phenotype. This protein (which has been determined to be the gene product of the MDR 1 gene in humans) facilitates active (i.e., energy-dependent) drug efflux from the cell, against a concentration gradient of (generally) lipophilic compounds, including many cytotoxic drugs.
The gene encoding P-glycoprotein (which is also known as gp 170-180 and the multidrug transporter) has been cloned from cultured human cells by Roninson et al. (see co-owned U.S. Pat. No. 5,206,352, issued Apr. 27, 1993, having an effective filing date of Mar. 28, 1986), and is generally referred to as MDR 1. The protein product of the MDR 1 gene, most generally known as P-glycoprotein ("Pgp"), is a 170-180 kilodalton (kDa) transmembrane protein having the aforementioned energy-dependent efflux pump activity.
Molecular analysis of the MDR1 gene indicates that Pgp consists of 1280 amino acids distributed between two homologous halves (having 43% sequence identity of amino acid residues), each half of the molecule comprising six hydrophobic transmembrane domains and an ATP binding site within a cytoplasmic loop. Only about 8% of the molecule is extracellular, and carbohydrate moieties (approximately 30 kDa) are bound to sites in this region (Chen et al., 1986, Cell 47: 381-387).
Expression of Pgp on the cell surface is sufficient to render cells resistant to many (but not all) cytotoxic drugs, including many anti-cancer agents. Pgp-mediated MDR appears to be an important clinical component of tumor resistance in tumors of different types, and MDR1 gene expression correlates with resistance to chemotherapy in different types of cancer.
Because Pgp is involved in the resistance of different types of human malignancies to conventional chemotherapy, the expression of Pgp is an important diagnostic and prognostic factor which in many cases helps the physician to choose the most effective combination of chemotherapeutic drugs and to monitor the efficacy of treatment. One way Pgp expression has been evaluated is by detecting the binding of specific immunological reagents (antibodies) to tumor samples. However, frequently the expression level of Pgp in tumor cells is low and cannot be reproducibly detected by routine immunological methods. In addition, there are few immunological or other reagents specific for functionally-active Pgp (which are the only forms of Pgp that are clinically relevant). Thus, there is a need in the art to increase the sensitivity and specificity of immunological and immunohistochemical methods for detecting functional Pgp expression.
Pgp is also constitutively expressed in many normal cells and tissues (see Cordon-Cardo et al., 1990, J. Histochem. Cytochem. 38: 1277; and Thiebaut et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7735 for reviews). In hematopoietic cells, Neyfakh et al. (1989, Exp. Cancer Res. 185: 496) have shown that certain subsets of human and murine lymphocytes efflux Rh123, a fluorescent dye that is a Pgp substrate, and this process can be blocked by small molecule inhibitors of Pgp. It has been demonstrated more recently that Pgp is expressed on the cell-surface membranes of pluripotent stem cells, NK cells, CD4- and CD8-positive T lymphocytes, and B lymphocytes (Chaudhary et al., 1992, Blood 80: 2735; Drach et al., 1992, Blood 80: 2729; Kimecki et al., 1994, Blood 83: 2451; Chaudhary et al., 1991, Cell 66: 85). Pgp expression on the cell surface membranes of different subsets of human lymphocytes has been extensively documented (Coon et al., 1991, Human Immunol. 32: 134; Tiirikainen et al., 1992, Ann. Hematol. 65: 124; Schluesener et al., 1992, Immunophannacology 23: 37; Gupta et al., 1993, J. Clin. Immunol. 13: 289). Although recent studies suggest that Pgp plays a role in normal physiological functions of immune cells (Witkowski et al., 1994, J. Immunol. 153: 658; Kobayashi et al., 1994, Biochem. Pharmacol. 48: 1641; Raghu et al., 1996, Exp. Hernatol. 24:1030-1036, as disclosed more fully in co-pending U.S. patent application, Ser. No. 08/658,583, filed Jun. 7, 1996, incorporated by reference herein in its entirety), the physiological role of Pgp in normal immune cells has remained unclear to date.
Expression of Pgp in hematopoietic cells provides an effective means for identifying and punfyng lymphocytes and hematopoietic stem cells. As described more completely in co-owned and/or co-pending U.S. Pat. No.5,434,075, issued Jul. 18, 1995 and U.S. Ser. No.08/032,056, filed Mar. 16, 1993, functional Pgp assays (such as fluorescent dye efflux) and immunochemical methods (such as fluorescence activated cell sorting (FACS) analysis) can in theory be used to purify lymphocytes and hematopoietic stem cells.
However, the levels of expression of Pgp on stem cells are low, and consequently the amount of an immunological reagent such as a monoclonal antibody (mAb) bound to a hematopoietic stem cell membrane using conventional procedures is generally not high enough to efficiently separate Pgp-positive cells by any conventional immunological technique (such as FACS, immunomagnetic particle separation, cell panning, or other methods known in the art). Thus, there remains a need in this art to improve the efficiency of methods for using Pgp expression to specifically purify lymphocytes and hematopoietic stem cells from biological sources.
Once the central role in MDR played by Pgp was uncovered, agents with a potential for reversing MDR phenotypes were developed that target Pgp. Several classes of drugs, including calcium channel blockers (e.g., verapamil), immunosuppresants (such as cyclosporines and steroid hormones), calnodulin inhibitors, and other compounds, were found to enhance the intracellular accumulation and cytotoxic action of Pgp-transported drugs (Ford et al., 1990, Pharm. Rev. 42: 155). Many of these agents were found to inhibit either drug binding or drug transport by Pgp (Akiyama et al., 1988, Molec. Pharm. 33: 144; Horio et al., 1988, Proc. Natl. Acad. Sci. USA 84: 3580). Some of these agents themselves were found to bind to and be effluxed by Pgp, suggesting that their enhancing effects on the cytotoxicity of Pgp substrates are due, at least in part, to competition for drug binding sites on this protein (Cornwell et al., 1986, J Bio.. Chem. 261: 792 1; Tamai, 1990, J. Biochem. Molec. Biol. 265: 16509).
Many of these agents, however, also have strong, deleterious side effects at physiologically-achievable concentrations. These systemic side effects severely limit the clinical use of these agents as specific inhibitors of Pgp or for negative selection against Pgp-expressing tumor cells. Most of the known MDR-reversing drugs used in clinical trials have major side effects unrelated to inhibition of Pgp, such as calcium channel blockage (verapamil) or immunosuppression (cyclosporines and steroids). Similarly, targeting of cytotoxic drugs to Pgp-expressing cells is capable of compromising normal tissue function in normal cells (such as kidney, liver, colonic epithelium, etc.) that normally express Pgp. These drawbacks restrict the clinically-achievable dose of such agents and ultimately, their usefulness.
Immunological reagents, specifically such agents linked to cytotoxic molecules or detectably labeled reporter molecules, provide an alternate and specific way for identifying cells expressing Pgp at the cell surface and specifically delivering cytotoxic substances directly to such cells. Immunological reagents specific for extracellular epitopes of Pgp, such as anti-Pgp antibodies, offered the prospect of specificity, since antibodies should target only Pgp. However, it has also been recognized that only antibodies which react with an extracellular epitope of Pgp are expected to react with the protein in the plasma membrane of intact cells and thereby inhibit the MDR phenotype in such cells. Antibodies directed to the cytoplasmic portion of Pgp, on the other hand, are unlikely to be useful for reversal of MDR.
In addition, antibody binding to Pgp was expected to have a more-prolonged inhibitory effect than that caused by transient binding of a competitive inhibitor. Such reagents may also provide a means for delivering cytotoxic agents specifically to Pgp-expressing tumor cells in regimens aimed to selective killing of such cells.
Monoclonal antibodies specific for Pgp are known in the art.
Hamada et al., 1986, Proc. Natl. Acad. Sci. USA 83: 7785 disclose the mAbs MRK-16 and MRK-17, produced by immunizing mice with doxorubicin-resistant K-562 human leukemia cells. MRK- 16 mAb was also reported to modulate vincristine and actinomycin D transport in resistant cells, and MRK-17 was shown to specifically inhibit growth of resistant cells with these drugs.
Meyers et al., 1987, Cancer Res. 49: 3209 disclose mAbs HYB-241 and HYB-612, which recognize an external epitope of Pgp.
O'Brien et al., 1989, Proc. Amer. Assoc. Cancer Res. 30:Abs 2114 disclose that mAbs HYB-241 and HYB-612 increased the accumulation of vincristine and actinomycin D in tumor cells and increased the cytotoxicity of combinations of these drugs with verapamil.
Tsuruo et al., 1989, Jpn. J Cancer Res. 80: 627 reported that treatment of athyrnic mice that had been previously inoculated with drug resistant human ovarian cancer cells with the mAb MRK 16 caused regression of established subcutaneous tumors.
Hamada et al., 1990, Cancer Res. 50: 3167 disclosed a recombinant chimeric antibody that combines the variable region of MRK-16 with the F.sub.c portion of a human antibody, and showed this chimeric antibody to be more effective than MRK-16 mAb in increasing cytotoxicity in vitro.
Pearson et al., 1991, J. Natl. Cancer Inst. 88: 1386 disclosed that MRK-16 mAb increased the in vivo toxicity of vincristine to a human MDR colon cancer cell line grown as a xenograft in nude mice. The in vitro potentiation of drug cytotoxicity by MRK-16 mAb was, however, weak relative to known chemical inhibitors of Pgp action, and was apparently limited to only two Pgp substrates (vincristine and actinomycin D), having no effect on cytotoxicity by doxorubicin.
Cinciarelli et al., 1991, Int. J. Cancer 47: 533 disclosed a mouse IgG.sub.2a mAb, termed MAb657, having cross reactivity to Pgp-expressing human MDR cells. This mAb was shown to increase the susceptibility of MDR cells to human peripheral blood lymphocyte-mediated cytotoxicity, but was not shown to have an inhibitory effect on the drug efflux activity of Pgp.
Arcesi et al., 1993, Cancer Res. 53: 310-317 disclosed mnAb 4E3 that binds to extracellular epitopes of Pgp but does not disrupt drug efflux or potentiate MDR drug-induced cytotoxicity.
Mechetner and Roninson, in co-owned and/or co-pending U.S. Pat. No. 5,434,075, issued Jul. 18, 1995, and in U.S. Ser. No. 08/032,056, filed Mar. 16, 1993, disclosed mAb UIC2, having specificity for extracellular Pgp epitopes. This antibody was also shown to effectively inhibit Pgp-mediated drug efflux in MDR cells, and to reverse the MDR phenotype in vitro thereby, for a number of structurally and functional different cytotoxic compounds and all tested chemotherapeutic drugs known to be substrates for Pgp-mediated drug efflux.
The production of UIC2 mAb demonstrated the usefulness of the development of mAbs specific for extracellular epitopes of Pgp that were capable of inhibiting drug efflux activity. As evidenced by the mAbs developed in the prior art, production of extracellular epitope-specific mAbs does not necessarily result in mAbs that can affect drug efflux. There thus remains in the art a need for developing methods for producing mAbs that are capable of inhibiting drug efflux activity in Pgp. There also remains a need in the art for methods for developing more sensitive mAbs and methods to improve the sensitivity of currently available mAbs for the detection of Pgp expression in cancer cells in vivo, for improved cancer diagnostics and therapeutic applications with both normal and tumor cells expressing Pgp. There also remains a need in the art to develop more specific and efficient tools for the isolation of lymphocytes and hematopoietic stem cells, especially pluripotent and totipotent stem cells.